DNA polymerase alpha mutants from a Drosophila melanogaster cell line.
Aphidicolin, a tetracyclic diterpenoid antibiotic, is a specific inhibitor of DNA synthesis in vivo and DNA polymerase (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) alpha of eukaryotic organisms. After ethyl methanesulfonate mutagenesis, we have recovered mutants of Drosophila melanogaster Schneider cell line no. 2 that grow at concentrations of aphidicolin that completely inhibit wild-type cells. The DNA polymerase alpha from one of these mutants, aph-10, is much more resistant to inhibition by the drug; the apparent Ki of the wild-type enzyme is 12 nM aphidicolin, whereas the apparent Ki of the aph-10 polymerase is more than 100 nM. (The apparent Km for dCTP is the same for both enzymes.) Another mutant, aph-13, overproduces DNA polymerase alpha at least 8-fold. The DNA polymerase of this mutant has the same apparent Km and Ki for dNTPs and aphidicolin as does wild-type polymerase.
D000818:Animals; D016590:Aphidicolin; D002460:Cell Line; D004261:DNA Replication / Q000187:drug effects; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D004224:Diterpenes / Q000494:pharmacology; D004331:Drosophila melanogaster / Q000201:enzymology / Q000235:genetics*; D004351:Drug Resistance; D005786:Gene Expression Regulation; D009154:Mutation; D019384:Nucleic Acid Synthesis Inhibitors
D004224:Diterpenes; D019384:Nucleic Acid Synthesis Inhibitors; D016590:Aphidicolin; D004259:DNA-Directed DNA Polymerase